Introduction

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Human BChE has a major role in detoxication of cocaine (Kalow and Grant, 2001). Treatment of rodents and cats with human or horse BChE protects from cocaine-induced hypertension, cardiac arrhythmia, hyperactivity, seizures, and lethality (Hoffman et al., 1996; Lynch et al., 1997; Mattes et al., 1997; Carmona et al., 1998). The amount of BChE present in human blood and tissues is insufficient to instantly detoxify inhaled or injected cocaine because BChE hydrolyzes the ()-cocaine isomer slowly. By contrast, the (+)-cocaine isomer is hydrolyzed 2000- fold faster (Gatley, 1991; Xie et al., 1999) and has none of the physiological effects of ()-cocaine. The lack of pharmacologic activity of (+)-cocaine is attributed to rapid hydrolysis by BChE (Gatley, 1991). A BChE with a faster rate of hydrolysis might render ()-cocaine pharmacologically inactive.

One purpose of this work was to increase the catalytic efficiency of human BChE for hydrolysis of ()-cocaine. A 4-fold increase in catalytic efficiency had been achieved with the A328Y mutant (Xie et al., 1999). In this report a further increase in catalytic efficiency is provided by the A328W mutant, which hydrolyzes ()-cocaine 15-fold faster than does wild-type BChE. The laboratory of Stephen Brimijoin has engineered other cocaine-hydrolyzing mutants of BChE (Sun et al., 2001). A large aromatic residue at position 328 is expected to orient ()-cocaine into position for attack by the active site serine (Xie et al., 1999).

A second goal was to produce the A328W mutant BChE in a form that would have a long residence time in the circulation of animals. Saxena et al. (1998) had shown that recombinant human BChE had a very short residence time in the circulation of mice, on the order of minutes, whereas native human BChE purified from plasma had a mean residence time of 46 h. A significant difference between recombinant and native BChE was that recombinant human BChE secreted by Chinese hamster ovary (CHO) cells consisted mainly of monomers and dimers (Blong et al., 1997; Saxena et al., 1998), whereas native BChE is a tetramer. The finding by Bon et al. (1997) and Krejci et al. (1997) that a proline-rich peptide from the N terminus of the collagen-tail protein (COLQ gene) caused assembly of acetylcholinesterase into tetramers led us to the experiments that solved the problem. Coexpression of BChE with a 45-amino acid peptide encoded by the COLQ gene converted 70% of the recombinant BChE to tetramers (Altamirano and Lockridge, 1999). Tetrameric recombinant BChE was found to have a residence half-time of 16 h in the circulation of rats and mice. This long half-time was achieved without modifying the carbohydrate content.

A third goal was to demonstrate the protective effect of recombinant human BChE against cocaine toxicity. Native wild-type BChE, purified from human or horse plasma, has previously been shown to protect mice, rats, and cats from cocaine toxicity (Hoffman et al., 1996; Lynch et al., 1997; Mattes et al., 1997; Carmona et al., 1998). However, recombinant human BChE has not previously been tested in animals. The protective effect of recombinant BChE tetramers was shown by measurement of locomotor activity in mice. Cocaine at a dose of 25 mg/kg i.p. induced high locomotor activity, but pretreatment with BChE reduced activity by 50 to 80%. The A328W mutant was more effective than wild-type BChE at reducing locomotor activity. These results indicate that recombinant wild-type and mutant BChE may be useful for treatment of cocaine toxicity in humans.

	Materials and Methods

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Mutagenesis. The mutation A328W was introduced into human BChE by polymerase chain reaction as previously described (Xie et al., 1999). The cloned cDNA was completely sequenced to verify the presence of the A328W mutation and the absence of unwanted mutations.

Expression of Human BChE. Human BChE was expressed in Chinese hamster ovary cells (CHO-K1; ATCC 61-CCL), stably transfected with plasmid pGS-BCHE wild-type or A328W as previously described (Xie et al., 1999). Selective pressure to retain the plasmid was provided by 50 µM methionine sulfoximine in the initial period and reduced to 25 µM for maintenance. Secreted BChE was collected into serum-free and glutamine-free culture medium, Ultraculture (BioWhittaker, Walkersville, MD, catalog number 12-725B), thus avoiding contamination by AChE present in fetal bovine serum. No antibiotics were added to the culture medium. The cells were grown in 1-liter roller bottles. Culture medium (150 ml per bottle) in the roller bottles was changed every 2 to 4 days. A roller bottle yielded BChE continuously for as long as 6 months. Each liter of culture medium contained 1 to 5 mg of BChE.

N Terminus of the Collagen-Tail. The proline-rich attachment domain is a 17-residue peptide from the N terminus of the collagen-tail encoded by the COLQ gene (Bon et al., 1997; Krejci et al., 1997). It has two cysteines as well as five and three consecutive prolines in the sequence CCLLMPPPPPLFPPPFF. Bon et al. (1997) first reported that this peptide caused recombinant AChE monomers to assemble into tetramers. A clone encoding 117 amino acids of the N terminus of the rat collagen-tail was a gift of Dr. Eric Krejci. We modified this clone by PCR. The modified collagen-tail was called rQ45; it included 22 codons for the signal peptide, 45 for the N terminus of COLQ, and 8 for the FLAG epitope DYKDDDDK cloned into the mammalian expression plasmid, pRc/RSV (Invitrogen, Carlsbad, CA) (see Fig. 1). The expression plasmid for rQ45 had a different promoter from the promoter for expression of BChE so that the two would not compete for transcription factors. The promoter for expression of rQ45 was Rous sarcoma virus long terminal repeat, whereas the promoter for expression of BChE was cytomegalovirus. The selectable marker in plasmid pRc/RSV-rQ45 is the NEO gene and in plasmid pGS is glutamine synthetase.

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Sequence of rQ45. The modified collagen-tail peptide was called rQ45; it included 22 codons for the signal peptide, 45 for the rat COLQ gene (Krejci et al., 1997), and 8 for the FLAG epitope DYKDDDDK cloned into the mammalian expression plasmid, pRc/RSV (Invitrogen) at the HindIII and NotI sites. Coexpression of rQ45 with BChE yields BChE tetramers.

Coexpression of Human BChE and rQ45. The tetramerization domain of human BChE is located at the C terminus where the 40 amino acids encoded by exon 4 are essential for formation of dimers and tetramers (Blong et al., 1997; Altamirano and Lockridge, 1999). Full-length BCHE cDNA encoding 574 amino acids of the mature protein was used for expression. Stable CHO cell lines expressing wild-type or A328W human BChE were transfected with pRc/RSV-rQ45. Clones expressing both BChE and rQ45 were selected in Ultraculture containing 25 µM methionine sulfoximine and 0.8 mg/ml G418 (geneticin). Cells were amplified in T150 flasks and finally in roller bottles for large-scale production of tetrameric BChE. After cells had coated the roller bottle, the G418 was no longer added to culture medium.

Purification of Recombinant BChE. Serum-free culture medium was collected from roller bottles over a period of months. The BChE-containing culture medium was stored at 4°C in sterile bottles. Ten to 20 liters of culture medium containing 50 to 100 mg of BChE were filtered through Whatman filter paper no. 1 (Whatman, Clifton, NJ) on a Buchner funnel, or through a coffee filter on a fritted glass funnel attached to a water aspirator, to remove cell debris. The filtered culture medium was loaded onto a 300- to 400-ml procainamide-Sepharose affinity column packed in a Pharmacia column XK50/30 (Pharmacia, Peapack, NJ). This column has a diameter of 5 cm, allowing a flow rate of 1 liter/h. All of the BChE activity was retained by the affinity gel. The column was washed with 20 mM potassium phosphate, 1 mM EDTA, pH 7, until the absorbance at 280 nm of the eluate was nearly zero. When the BChE on the column was wild-type BChE, the column was washed with 0.2 M NaCl in buffer to elute contaminating proteins. When the BChE was A328W, the column was washed with 0.6 M NaCl in buffer because A328W remained bound to the affinity gel at this salt concentration. The column was washed with buffer before eluting wild-type BChE with 1 M NaCl or A328W with 2 M NaCl containing 0.2 M choline chloride in 20 mM potassium phosphate, pH 7. The yield of BChE from this first step was 90 to 100%.

Purification of BChE from Human Plasma. Native BChE was purified from human plasma by ion-exchange chromatography at pH 4.0, followed by affinity chromatography on procainamide Sepharose.

Affinity Gel. Procainamide-Sepharose 4B affinity gel, with a 6 carbon spacer, was custom made by Yacov Ashani at the Israel Institute for Biological Research, Ness-Ziona, Israel. The concentration of bound procainamide was estimated to be 34 µmol/ml. Used affinity gel was recycled by washing on a fritted glass funnel with 0.5 M glacial acetic acid, followed by water. The washed gel was stored in the presence of 20% ethanol at 4°C. The gel has been reused repeatedly for several years, with no apparent loss in binding capacity.

k_cat and K_m for Cocaine, Butyrylthiocholine, and Benzoylcholine. ()-Cocaine hydrochloride (purchased from Sigma-Aldrich, St. Louis, MO, after obtaining a controlled substance license from the U.S. Department of Justice) was dissolved in water to make a 0.1 M stock containing 34 mg/ml. Aliquots were frozen at 80°C, thawed once, and discarded. The rate of hydrolysis of ()-cocaine was measured in the spectrophotometer at 240 nm, using an extinction coefficient of 6700 M¹ cm¹ for the difference in absorbance between cocaine and benzoic acid (Gatley, 1991). The temperature was 25°C, and the buffer was 0.1 M potassium phosphate, pH 7.0.

BChE Activity Assay. Serum samples were tested for BChE activity with 1 mM butyrylthiocholine and 0.5 mM 5,5'-dithio-bis-(2-nitrobenzoic acid), in 0.1 M potassium phosphate buffer, pH 7.0, at 25°C. A temperature-controlled Gilford spectrophotometer that interfaced via a MacLab data recorder (ADInstruments Pty Ltd., Castle Hill, Australia) to a Macintosh computer (Apple, Cupertino, CA) was used. Formation of the product was followed by the absorbance increase of 5-thio-2-nitrobenzoic acid at 412 nm, using a molar extinction coefficient of 13,600 M¹ cm¹ (Ellman et al., 1961). Activity is reported as units per milliliter, where 1 unit represents the hydrolysis of 1 µmol of butyrylthiocholine per min.

Nondenaturing Gel Electrophoresis The relative amount of tetramers, dimers, and monomers was estimated on activity-stained nondenaturing polyacrylamide gels. Four to 30% polyacrylamide gradient gels were prepared in a Hoeffer SE600 gel apparatus (Hoeffer, San Francisco, CA; presently part of Pharmacia, Inc.). Electrophoresis was at 120 V constant voltage for 15 h at 4°C. It was important to keep the gels cold during electrophoresis to avoid degrading the heat-labile monomers and dimers (Blong et al., 1997). Gels were stained for BChE activity in the presence of 2 mM butyrylthiocholine iodide by the method of Karnovsky and Roots (1964). Band intensity was quantified with a BioImage 110S System (Millipore).

Elimination Time of Human BChE from Rats and Mice. Animal studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the U.S. National Institutes of Health. Five male Sprague-Dawley rats weighing approximately 350 g were anesthetized by intraperitoneal administration of 60 mg/kg -chloralose and 800 mg/kg urethane. A cannula made of Silastic tubing was implanted into the jugular vein. Blood for the zero time point was collected. Human BChE was delivered through the cannula to a dose of 1 mg/kg; that is, 0.35 ml of 1 mg/ml BChE (720 U/ml for wild-type BChE) was infused over a 45-s period. The cannula was flushed with 0.1 ml of saline, and a blood sample was collected 1 min after the flush with saline. Blood samples at 1, 3, 6, 12, 24, and 36 h were also collected through the cannula. The rat serum was tested for BChE activity.

Locomotor Activity. A motion detector was made by the Instrument Shop at the University of Nebraska Medical Center. It consisted of a red-light-emitting diode that sent a beam of light through the cage wall, a photodiode detector on the opposite side of the cage, and a microprocessor. When the mouse passed through the beam, an event was recorded by the microprocessor. After a defined time, the total number of events was printed. The beam was set up in a dimly lit box containing the home cage in a sound-proof room. The cage contained no bedding because kicked-up bedding could trigger the beam counter. The mouse was acclimated for 1 h before beam breaks were counted.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Catalytic Activity of A328W. The A328W mutant of BChE was tested for activity with ()-cocaine, butyrylthiocholine, and benzoylcholine. Table 1 shows that A328W hydrolyzed ()-cocaine 15-fold faster than did wild-type BChE as measured by k_cat. The binding affinity was nearly the same for both enzymes, the K_m value being 10 µM for A328W and 7 µM for wild-type BChE. Both enzymes hydrolyzed butyrylthiocholine and benzoylcholine much more rapidly than cocaine. However, the k_cat value for A328W was lower for these substrates compared with the k_cat for wild-type BChE.

Tetramers of Recombinant BChE. When BChE was expressed in stable CHO cell lines in the absence of the peptide from COLQ, the BChE consisted predominantly of dimers and monomers, as demonstrated on nondenaturing gel stained for BChE activity. Only 10% of the BChE activity was a tetramer (Blong et al., 1997; Altamirano and Lockridge, 1999). However, coexpression of 45 residues of the N terminus of the rat collagen-tail, called rQ45, resulted in 70% BChE tetramers (Fig. 2).

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Nondenaturing gel stained for BChE activity. Recombinant human BChE secreted from CHO cells consists mainly of monomers and dimers. Recombinant human BChE rQ45 consists of 70% tetramers. Human plasma contains more than 95% tetramers. The plasma is a control to show the migration of monomers, dimers, and tetramers of BChE. The C2 band is a dimer made by disulfide bonding of albumin and BChE (Masson, 1989).

Elimination Half-Life in Rats. When a purified preparation of recombinant human BChE monomers and dimers was injected into the jugular vein of rats, the half-life was 2 min (Fig. 3), a result in agreement with that of Saxena et al. (1998). No detectable human BChE was present in rat serum after 1 h. In contrast, when a preparation containing 70% tetramers (wild-type BChE rQ45 or A328W rQ45) was injected into rats, the elimination was biphasic, with 81% disappearing with a half-life of 26 ± 3 min and 19% disappearing with a half-life of about 16 ± 6 h (Fig. 3). The mean residence time was 1245 min. Significant amounts of human BChE activity were still present in the rat 24 h after i.v. injection of tetrameric BChE. The 1 to 2.8 U/ml of BChE activity found after 24 h is a 40- to 50-fold increase above the endogenous BChE activity of 0.02 to 0.07 U/ml in rat serum.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Elimination half-life of recombinant BChE injected i.v. into rats. Purified recombinant wild-type BChE consisting of either 70% tetramers or 90 to 95% monomers and dimers was injected intravenously into rats (n = 5) at a dose of 1 mg/kg (250 units per rat). The same results were obtained with recombinant A328W rQ45.

View larger version (67K): [in this window] [in a new window]   Fig. 4.   Nondenaturing gradient polyacrylamide gel stained for BChE activity in rat. Blood samples were removed at various times after treatment of a rat with purified recombinant wild-type human BChE rQ45. Two microliters of serum were placed in each well of a 4 to 30% gradient polyacrylamide gel and electrophoresed, as described under Materials and Methods. Tetramers of human BChE were still present after 24 to 36 h, although dimers and monomers had disappeared. Human serum provides markers for tetramers, dimers, and monomers.

Peak Activity in Blood. Five mice received intraperitoneal injections of the same purified BChE preparation (recombinant wild-type human BChE rQ45 containing 70% tetramers) that had been given to rats. Figure 5 shows that the human BChE rapidly entered mouse blood, where peak activity was achieved about 1 h after injection into the peritoneal cavity. Peak activity in mouse blood was 45-fold above the endogenous activity of 1 to 1.4 U/ml. The activity remained high for hours. After 7 h, it was still 20-fold over endogenous activity. Hoffman et al. (1996) had previously found that native human BChE injected i.p. reached peak activity in mouse blood 1 h after injection. Based on these results, we decided to allow 1 h to elapse between i.p. injection of BChE and injection of cocaine in locomotor activity assays.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   BChE activity in mouse blood after intraperitoneal injection. Purified wild-type BChE rQ45 consisting of 70% tetramers was injected intraperitoneally into mice (n = 5) at a dose of 9 mg/kg.

Elimination Half-Life in Mice for Recombinant BChE. Figure 6 shows that recombinant BChE disappeared from mouse blood in a biphasic manner, with 57% disappearing with a half-life of 48 ± 20 min and 43% with a half-life of 15.6 ± 2 h. The mean residence time was 1269 min. The pharmacokinetics of the purified human BChE rQ45 were similar in rats and mice.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Elimination half-life of recombinant human BChE injected i.p. into mice. Purified wild-type human BChE rQ45 consisting of 70% tetramers was injected intraperitoneally into mice (n = 5) at a dose of 9 mg/kg.

View larger version (53K): [in this window] [in a new window]   Fig. 7.   Nondenaturing polyacrylamide gel stained for BChE activity in mouse. Blood samples were removed from a mouse that was treated intraperitoneally with purified recombinant wild-type human BChE rQ45. Two microliters of mouse serum were placed in each well.

Elimination Half-Life in Mice for Native Human BChE. Figure 8 shows the BChE activity in mouse blood after an i.p. injection of 438 units of native human BChE tetramers (0.6 mg of BChE per mouse). High amounts of human BChE were present in mouse blood 1 to 10 h after injection. The mean residence time was 56.6 h. These results show that the residence time of native human BChE in the mouse circulation is 2.7-fold longer than the residence time of recombinant human BChE.

View larger version (31K): [in this window] [in a new window]   Fig. 8.   Elimination half-life of native human BChE injected i.p. into mice. Purified native human BChE consisting of 99% tetramers was injected intraperitoneally into mice (n = 5) at a dose of 27 mg/kg.

Cocaine Toxicity Measured as Locomotor Activity. A dose of 25 mg/kg cocaine i.p. caused increased locomotor activity in mice. Their behavior was markedly different from that of control mice (n = 32) injected with saline or BChE alone, who ambled back and forth for a few minutes, sniffed, defecated, urinated, then settled down and went to sleep (Fig. 9). Locomotor hyperactivity was therefore taken to be a good indicator of cocaine toxicity.

View larger version (45K): [in this window] [in a new window]   Fig. 9.   Locomotor activity in mice as a measure of cocaine toxicity. Mice were acclimated for 1 h before receiving 25 mg/kg cocaine i.p. (n = 10). Another group of mice (n = 10) was acclimated for 1 h, then received 2.8 units/g of wild-type BChE rQ45 i.p., followed in 60 min by an injection of 25 mg/kg cocaine i.p. A third group (n = 10) was pretreated with 2.8 units/g of A328W rQ45 before receiving cocaine. A fourth group (n = 15) was pretreated with 2.8 units/g of native human BChE before receiving cocaine. Locomotor activity was recorded for 60 min following BChE injection and 60 min after cocaine injection. The A328W mutant of BChE was more effective than wild-type BChE at attenuating locomotor activity induced by cocaine.

	Discussion

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Protection against Cocaine Toxicity. Both wild-type recombinant human BChE and the A328W mutant BChE protected mice from cocaine-induced hyperactivity. The A328W mutant gave more protection, consistent with its higher catalytic activity toward cocaine. Native human wild-type BChE, purified from human plasma, gave a level of protection similar to that of recombinant wild-type human BChE.

Recombinant BChE with a 16-h Half-Life in the Circulation. This is the first report in which recombinant human BChE has been used in protection experiments. Previous work has used native BChE purified from plasma for protection against cocaine toxicity (Hoffman et al., 1996; Lynch et al., 1997; Mattes et al., 1997; Carmona et al., 1998) and for protection against organophosphorus toxicants (Broomfield et al., 1991; Doctor et al., 1991; Brandeis et al., 1993; Raveh et al., 1997).

Carbohydrate Content of Recombinant BChE. Saxena et al. (1998) found that recombinant human BChE monomers and dimers secreted by CHO cells are underglycosylated. Recombinant BChE has only five N-glycans, whereas native human BChE has nine. The recombinant BChE had a ratio of sialic acid to galactose of about 1, suggesting that nearly all galactose residues were capped with sialic acid. Saxena et al. (1998) concluded that the capping of galactose with sialic acid by itself is not sufficient to confer circulatory stability and that high molecular weight is also important. Our results support the conclusion that the rapid clearance of BChE monomers and dimers is not due to incomplete sialylation but to their small size of 85 and 170 kDa. It was not necessary to modify the carbohydrate content of the recombinant BChE to attain a half-life of 16 h. Assembly into tetramers achieved the desired goal. This contrasts with recombinant AChE expressed in human embryonic kidney 293 cells (Kronman et al., 2000), where a half-life of 15 h was obtained only after modification of the carbohydrate content and after assembly into tetramers. The importance of large molecular size was confirmed by Cohen et al. (2001), who attached polyethylene glycol to AChE monomers and found that high molecular weight monomers had a half-life of 26 h in the circulation of mice, even though only 60% of the N-glycans were sialylated.

CHO Cells. CHO cells are the preferred cell type for expression of human glycoproteins of potential therapeutic value such as erythropoietin, human growth factor, and tissue plasminogen activator (Jenkins and Curling, 1994). Our decision to produce recombinant human BChE in CHO cells was made after we compared growth conditions and yield of BChE in CHO cells and in 293 human embryonic kidney cells. The 293 cells are used to produce human AChE (Kronman et al., 2000). We found that the yield of human BChE was similar in both cell lines. Both cell lines secreted human BChE to a maximum of 5 mg/l. Both cell lines produced monomers and dimers and very few tetramers. Kronman et al. (2000) had to modify the 293 cells to produce 2,6-sialyltransferase, an enzyme deficient in 293 cells, to fully sialylate the AChE. Although CHO cells are also deficient in 2,6-sialyltransferase (Jenkins and Curling, 1994), the recombinant BChE produced by CHO cells was fully sialylated (Saxena et al., 1998), and the tetrameric BChE produced by CHO cells had a half-life of 16 h without introducing sialyltransferase. An additional advantage of CHO cells is their ability to grow in the absence of fetal bovine serum.

Purification of Recombinant BChE. Recombinant BChE secreted by CHO cells was collected over a period of months. The culture medium was stored at 4°C until 100 mg of BChE had accumulated. No loss of BChE activity resulted during the storage period. A two-step purification procedure consisting of affinity chromatography on procainamide-Sepharose followed by ion-exchange chromatography on DE52 resulted in highly purified BChE tetramers. The simplicity of the purification protocol makes it possible to produce gram quantities of highly purified recombinant BChE.

Potential for Use of Human BChE in People. Purified BChE has a history of use in the clinic. The literature reports 134 patients who received partially purified human BChE for treatment of prolonged neuromuscular block by the muscle relaxants succinylcholine and mivacurium or for treatment of organophosphorus pesticide poisoning (Table 3). The BChE injected into these patients was a 5% pure preparation from human plasma sold by Behringwerke (Marburg, Germany). It is a dry concentrate of "cholase". The patients recovered and had no side-effects from the BChE treatment. All of the reports in Table 3 are from Europe or Saudi Arabia, as human BChE is not yet approved for human use in the United States.